The expression of heterologous proteins represents a cornerstone of the biotechnology enterprise. Unfortunately, many commercially important proteins misfold and aggregate when expressed in a heterologous host (See, e.g., Makrides, Microbiol Rev 60, 512-538 (1996); Baneyx and Mujacic, Nat Biotechnol 22, 1399-1408 (2004); Georgiou and Valax, Curr Opin Biotechnol 7, 190-197 (1996)). Similarly, protein misfolding and aggregation is the pathological hallmark of more than a dozen diseases including Alzheimer's (See, e.g., Radford et al., Cell 97, 291-298 (1999); Ross and Poirier, Nat Med 10 Suppl, S10-17 (2004)). As if this weren't enough, existing biochemical means for assessing the tendency of proteins to misfold and aggregate are tedious. As a result, screening for constructs and/or conditions that favor solubility is inefficient and genetic selection of folded structures has not been forthcoming.
Development of a robust assay for in vivo protein folding and solubility has been challenging for researchers because of limitations on detecting and reporting the solubility of a protein. Existing systems for monitoring protein misfolding in vivo have capitalized on the observation that a misfolded target protein will often co-translationally induce improper folding of a C-terminally fused reporter protein (See, e.g., Maxwell et al., Protein Sci 8, 1908-1911 (1999); Waldo et al., Nat Biotechnol 17, 691-695 (1999)) or protein fragment (See, e.g., Cabantous et al., Nat Biotechnol 23, 102-107 (2005); Wigley et al., Nat Biotechnol 19, 131-136 (2001)) or will induce a specific gene response (See, e.g., Lesley et al., Protein Eng 15, 153-160 (2002)). This fusion approach is often problematic as certain reporter proteins can remain active even when the target protein to which they are fused aggregates or forms inclusion bodies (See, e.g., Tsumoto et al., Biochem Biophys Res Commun 312, 1383-1386 (2003)) while the gene expression response is limited by its indirect connection to the folding process.
Additionally, existing assays for protein expression in soluble form are tedious, usually requiring lysis and fractionation of cells followed by protein analysis by SDS-polyacrylamide gel electrophoresis. Using these traditional approaches, screening for protein constructs and/or physiological conditions yielding improved solubility is inefficient, and genetic selection nearly impossible.
Thus, there remains a need for new compositions and methods (e.g., assays) for monitoring, altering and/or selecting folded and soluble proteins (e.g., in vivo or in vitro). Such methods and compositions should be able to rapidly improve the soluble yield of a target protein by optimizing its primary sequence (e.g., through genetic selection) (See, e.g., Roodveldt et al., Curr Opin Struct Biol 15, 50-56 (2005)) or its cellular folding environment (See, e.g., Wall and Pluckthun, Curr Opin Biotechnol 6, 507-516 (1995)). Furthermore, such methods and compositions should be readily amenable to assay for agents (e.g., pharmaceuticals, drugs, small molecules, etc.) that either promote the folding/inhibit the aggregation of proteins associated with human disease (e.g. Alzheimer's Aβ42 peptide) (See, e.g., Williams et al., Proc Natl Acad Sci USA (2005)), or, on the contrary, agents that alter proper folding and induce aggregate formation (e.g., that could be used as antibiotics).